Compounds for treating corona virus infection

ABSTRACT

Described herein are compounds of Formula I:wherein R1-R6 are as described herein, for use in the treatment of a coronavirus infection; a method of inhibiting a coronavirus 3CL protease, by contacting the 3CL protease with a compound of Formula I; as well as methods pharmaceutical composition comprising a compound of Formula I and at least one phospholipid, wherein a weight ratio of the phospholipid(s) to the compound in the composition is in a range of from 10:1 to 1:10. Further described herein is a method of treating a coronavirus infection in a subject in need thereof, by administering to the subject at least one compound that exhibits at least two of: inhibition of an activity of a 3CL protease of the coronavirus; inhibition of inflammation in the subject; and inhibition of autophagy in the subject.

RELATED APPLICATIONS

This application is a Continuation of PCT Patent Application No.PCT/IB2021/051256 having International filing date of Feb. 15, 2021,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 62/976,420 filed on Feb. 14, 2020.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapy,and more particularly, but not exclusively, to compounds andcompositions useful for treating coronavirus-associated diseases, suchas COVID-19.

Coronaviruses are divided into alphacoronaviruses and betacoronaviruses(which infect mammals) and gammacoronaviruses and deltacoronaviruses(which primarily infect birds). The alphacoronaviruses include humancoronaviruses 229E and NL63 (two of the many viruses associated with thecommon cold and other respiratory tract infections); whereasbetacoronaviruses include human coronaviruses OC43 and HKU1 (additionalviruses associated with the common cold and other respiratory tractinfections), as well as Middle East respiratory syndrome-relatedcoronavirus and Severe acute respiratory syndrome (SARS)-relatedcoronaviruses such as SARS-CoV-1 (which is associated with the 2002-2004SARS outbreak) and SARS-CoV-2 (which is associated with COVID-19).

Coronaviruses belong to the nidovirus order, part of thepicornavirus-like supercluster, which includes picornaviruses,caliciviruses, and coronaviruses. These viruses possess a 3C protease(3Cpro) or 3C-like (3CL) protease (3CLpro), a cysteine protease whichgenerally contains a typical chymotrypsin-like fold and a catalytictriad or dyad comprising a cysteine residue and a nucleophile. Theseproteases cleave the viral polyprotein, which is essential for viralreplication.

3CLpro has been identified in several studies as an effective drugtarget for treating coronavirus infection [Yang et al., Curr Pharm Des2006, 12:4573-4590]. The 3CLpro of the pandemic-causing SARS, MERS and2019 Wuhan coronaviruses exhibits a high degree of conservation; forexample, the homology between the 3CLpro of SARS and 2019 Wuhancoronavirus is 98%.

“Shikonin” refers to5,8-dihydroxy-2-(1-hydroxy-4-methylpent-3-en-1-yl)napthalene-1,4-dione;and more particularly to the (R)-enantiomer thereof, which is found inthe roots of Lithospermum erythrorhizon (purple gromwell), a plant usedin Chinese herbal medicine (in which it is referred to as “zicao”) fortreating a variety of inflammatory and infectious diseases. The(S)-enantiomer is known as “alkannin”, a plant which has been used infolk medicine to treat abscesses and inflammations; and racemic mixturesare also known as “shikalkin”.

Shikonin has been reported to exhibit activities related to treatment ofcancer, inflammation and wound healing [Guo et al., Pharmacol Res 2019,149:104463]; as well as antiviral activity against HIV type I,adenovirus 3 (AdV3) and hepatitis C virus (HCV); and distinctanti-inflammation mechanisms, such as inhibition of leukotriene B₄synthesis, suppression of mast cell degranulation, inhibition ofneutrophil respiratory burst, alteration ofphosphatidylinositol-mediated signaling, and blockade of chemokinebinding to CCR-1 [Andujar et al., Planta Med 2013, 79:1685;] andreduction of interleukin-6, interleukin-8 and chemokine C—C motif ligand(CCL)20 production [Shindo et al., Inflammation 2016, 39:1124-1129].

U.S. Patent Application Publication No. 2004/0198716 describes quinonesand quinone analogs, including shikonin and alkannin, useful forinhibiting cysteine proteases such as caspases and 3C cysteineproteases.

U.S. Patent Application Publication No. 2008/0182900 describes a use ofcompounds such as shikonin and alkannin in treating infections by avirus (such as hepatitis virus, influenza virus, herpes virus, HIV andSARS virus) or mycoplasma, and malignant tumors.

Jo et al. [Chem Biol Drug Des 2019, 94:2023-2030] describes inhibitionof MERS coronavirus 3CLpro by herbacetin, isobavachalcone, quercetin3-β-D-glucoside, and helichrysetin; and concludes that flavonol andchalcone are suitable scaffolds for binding with the catalytic site ofMERS coronavirus 3CLpro.

Lu et al. [Asian J Pharm Sci 2019, 14:265-274] describes enhancement ofabsorption of active agents extracted from plants, by formation ofcomplexes of the active agent with amphipathic phospholipids at definedmolar ratios (described therein as “phyto-phospholipid complexes” or“phytosomes”).

Coronaviruses further comprise a papain-like (PL) protease, which isresponsible for cleaving the viral polyprotein at certain positionswhich are not cleaved by 3CL protease [Pillaiyar et al., Med Chem 2015,5:361-372]. In addition, some coronaviruses use activation by TMPRSS2(Transmembrane protease, serine 2), a human serine protease, to entercells. Inhibitors of PL protease and TMPRSS2 have been proposed forantiviral use.

Drugs which have been used to treat coronavirus infection includechloroquine and hydroxychloroquine. These drugs have been reported toexhibit moderate antiviral activity against a wide variety of viruses,which may be associated with inhibition of autophagy, a natural cellprocess which many viruses use to promote their own replication.

Additional background art includes U.S. Patent Application PublicationNo. 2005/0222258; Assimopoulou et al. [Biomed Chromatogr 2009,23:182-198]; Chen et al. [Phytother Res 2002, 16:199-209]; Dai et al.[Science 2020, 368:1331-1335]; Fan et al. [Pharm Biol 2018, 56:415-421];Lu et al. [Eur J Pharmacol 2011, 658:242-247]; Papageorgiou et al. [CurrMed Chem 2008, 15:3248-3267]; Ramajayam et al. [Bioorg Med Chem Lett2010, 20:3569-3572]; Xia et al. [Biosci Rep 2013, 33:e00020]; Xiong etal. [J Ethnopharmacol 2013, 145:573-580]; and Zhu et al. [ACS PharmacolTransl Sci 2020, 3:1008-1016].

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the invention, there isprovided a compound for use in the treatment of a coronavirus infection,the compound being represented by Formula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring.

According to an aspect of some embodiments of the invention, there isprovided a pharmaceutical composition comprising:

a compound represented by Formula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring; and

at least one phospholipid,

wherein a weight ratio of the at least one phospholipid to the compoundin the composition is in a range of from 10:1 to 1:10.

According to an aspect of some embodiments of the invention, there isprovided a method of inhibiting a coronavirus 3CL protease, the methodcomprising contacting the 3CL protease with a compound represented byFormula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring.

According to an aspect of some embodiments of the invention, there isprovided a method of treating a coronavirus infection in a subject inneed thereof, the method comprising administering to the subject atleast one compound that exhibits at least two of:

(i) inhibition of an activity of a 3CL protease of the coronavirus;

(ii) inhibition of inflammation in the subject; and

(iii) inhibition of autophagy in the subject,

thereby treating the coronavirus infection.

According to some embodiments of any of the embodiments relating to amethod of treating a coronavirus infection by administering a compound,the compound is represented by Formula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring.

According to some embodiments of any of the embodiments relating toFormula I, at least one of R₃ and R₆ is OH.

According to some embodiments of any of the embodiments relating toFormula I, R₃ and R₆ are each OH.

According to some embodiments of any of the embodiments relating toFormula I, R₂, R₄ and R₅ are hydrogen.

According to some embodiments of any of the embodiments relating toFormula I, R₁ is represented by:

wherein R′₁, R″₁, and R₇ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heteroalicyclic, and carbonyl.

According to some embodiments of any of the respective embodiments, R′₁is alkenyl.

According to some embodiments of any of the respective embodiments, R′₁is —CH₂—CH═C(CH₃)₂.

According to some embodiments of any of the respective embodiments, R₇is a saccharide moiety (e.g., glycosyl) or a peptide moiety.

According to some embodiments of any of the respective embodiments, R₇is hydrogen or carbonyl.

According to some embodiments of any of the embodiments described hereinrelating to a compound, the compound is shikonin or a glycoside ofshikonin, or an ester thereof.

According to some embodiments of any of the embodiments described hereinrelating to a compound or a method utilizing the compound, the compoundis part of a composition which further comprises one or morephospholipid(s), and a weight ratio of the phospholipid(s) to thecompound in the composition is in a range of from 10:1 to 1:10.

According to some embodiments of any of the embodiments described hereinrelating to a compound or a method utilizing the compound, the compoundis part of a composition which further comprises liposomes.

According to some embodiments of any of the embodiments described hereinrelating to a composition, the composition further comprising liposomes.

According to some embodiments of any of the embodiments described hereinrelating to a composition, the composition further comprises at leastone additional active agent selected from the group consisting of avitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatoryagent, an antipyretic agent, an antiviral agent, and a proteaseinhibitor.

According to some embodiments of any of the embodiments described hereinrelating to a composition, the composition is for use in the treatmentof a coronavirus infection.

According to some embodiments of any of the embodiments described hereinrelating to a method of inhibiting a coronavirus 3CL protease, themethod comprises administering a compound described herein to a subjectin need thereof.

According to some embodiments of any of the embodiments described hereinrelating to a treatment, the treatment is of a subject in whichinhibiting inflammation would be beneficial.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administration of thecompound from 2 to 6 times per day.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises intravenous administrationof the compound.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administering the compoundat a dose in a range of from 0.04 mg to 400 mg.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises oral administration of thecompound.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administering the compoundat a dose in a range of from 10 mg to 5 grams.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administering the compoundat a dosage in a range of from 2 mg per kg per day to 200 mg per kg perday.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administering at least oneadditional active agent selected from the group consisting of a vitamin,N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, anantipyretic agent, an antiviral agent, and a protease inhibitor.

According to some embodiments of any of the respective embodimentsdescribed herein, a dosage of N-acetyl cysteine is in a range of from0.2 to 6.4 grams per day.

According to some embodiments of any of the embodiments described hereinrelating to an additional active agent which is a protease inhibitor,the protease inhibitor is capable of inhibiting 3C protease and/or a 3CLprotease.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method comprises administering the at leastone additional active agent 4 times per day.

According to some embodiments of any of the embodiments described hereinrelating to a treatment or a method of inhibiting a coronavirus 3CLprotease, the treatment or method further comprises inhibitinginflammation and/or autophagy in the subject.

According to some embodiments of any of the embodiments relating to amethod of treating a coronavirus infection, the method comprisesadministering to the subject a compound capable of at least two ofinhibition of activity of 3CL protease, inhibition of inflammation, andinhibition of autophagy.

According to some embodiments of any of the embodiments relating to amethod of treating a coronavirus infection, the compound is capable ofeach of inhibition of activity of 3CL protease, inhibition ofinflammation, and inhibition of autophagy.

According to some embodiments of any of the embodiments relating to amethod of treating a coronavirus infection, the method comprisesadministering to the subject the compound of Formula I (according to anyof the respective embodiments described herein) and at least oneadditional agent that exhibits inhibition of activity of 3CL protease,inhibition of inflammation, and/or inhibition of autophagy.

According to some embodiments of any of the embodiments described hereinrelating to a coronavirus, the coronavirus is a betacoronavirus.

According to some embodiments of any of the embodiments described hereinrelating to a coronavirus, the coronavirus is a SARS-relatedcoronavirus.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A and 1B present exemplary naphthoquinone compounds according tosome embodiments of the invention.

FIG. 2 presents a bar graph showing the average of the maximal values oflevels of CRP, fibrinogen, D-dimer, troponin (trop) and ferritin inCOVID-19 patients treated with an composition comprising purple gromwellextract (Group A) or with vitamin C and spirulina dietary supplement(Group B); Group A included 10 patients and Group B included 19patients.

FIG. 3 depicts exemplary additional active agents which may beco-administered with a 3CL protease inhibitor according to someembodiments of the invention.

FIG. 4 presents a photograph of exemplary enteric-coated shikonintablets according to some embodiments of the invention.

FIG. 5 presents a graph showing release of shikonin from exemplaryenteric-coated shikonin tablets as a function of time, wherein the first120 minutes is under simulated gastric conditions and the next 120minutes are under simulated intestinal conditions.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapy,and more particularly, but not exclusively, to compounds andcompositions useful for treating coronavirus-associated diseases, suchas COVID-19.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

In a search for treatments of coronavirus infection with an acceptablelevel of side effects, the present inventor has uncovered a class ofnaphthoquinone compound, including many naturally occurring compounds,which exhibit a surprising ability to treat coronavirus infections, forexample, by exhibiting an antiviral effect and/or an anti-inflammatoryeffect.

While reducing the present invention to practice, the present inventorhas shown the efficacy of shikonin at inhibiting 3CL protease ofSARS-coronavirus 2, and the ability of plant extracts containingshikonin derivatives to treat SARS-coronavirus 2 infection.

As shown in the Examples section herein, exemplary compositionscomprising purple gromwell extract, known to contain shikonin orderivatives thereof, reduced mortality, signs of inflammation and otherdisease symptoms in patients afflicted by COVID-19, relative to acontrol treatment. As further shown therein, such compositions exhibitedinhibition of viral 3CL protease.

Thus, according to an aspect of some embodiments of the invention thereis provided a compound, as described herein, which may useful in thetreatment of a viral infection, for example, a coronavirus infection.

Compound:

The compound according to some of any of the respective embodimentsdescribed herein is represented by Formula I:

wherein R₁-R₆ are each independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl,sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl,carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl,hydrazine, hydrazide, thiohydrazide, and/or amino, or alternatively, R₁and R₂ together form a five- or six-membered aromatic or aliphatic ring.

For the sake of brevity, a compound according to Formula I is referredto herein interchangeably as a “naphthoquinone compound”.

In some of any of the respective embodiments described herein, at leastone of R₃ and R₆ is OH. In some such embodiments, R₃ and R₆ are each OH.In some embodiments, R₃ and R₆ are each OH and R₂, R₄ and R₅ are eachhydrogen.

Alternatively, at least one of R₃ and R₆ (and optionally both R₃ and R₆)is a functional group derived from OH (rather than OH), such asO-carboxy or a saccharide moiety (which may form OH upon cleavage of theester bond).

In some of any of the respective embodiments described herein, each ofR₃-R₆ is each independently OH (or a functional group derived from OH)or hydrogen.

In some of any of the respective embodiments described herein, R₂, R₄and R₅ are each hydrogen.

In embodiments, wherein R₁ and R₂ together form a five- or six-memberedaromatic or aliphatic ring, the ring may be unsubstituted orsubstituted, e.g., by any substituent described herein for a cycloalkyl,heteroalicyclic, aryl or heteroaryl. In some such embodiments, the ringis an aromatic carbon ring, such that the compound is a substituted orunsubstituted 9,10-anthroquinone. Exemplary 9,10-anthroquinones aredepicted in FIG. 1B.

In some of any of the respective embodiments described herein, R₁ is asubstituted or unsubstituted alkyl or alkenyl. In some such embodiments,R₂ is hydrogen.

In some of any of the respective embodiments described herein, R₁ isrepresented by:

wherein R′₁, R″₁, and R₇ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heteroalicyclic, and carbonyl. By “and/or” it is meant that the compoundmay comprise one stereoisomer or the other, or a racemic mixture inwhich both stereoisomer are present in approximately equal proportions,or a mixture enriched with stereoisomer relative to the other.

In some of any of the respective embodiments described herein, R′₁ isalkenyl, for example, —CH₂—CH═C(CH₃)₂.

In some of any of the respective embodiments described herein, R″₁ ishydrogen.

In some of any of the respective embodiments described herein, R₇ ishydrogen (e.g., as in shikonin) or a carbonyl group (e.g., as in anester of shikonin), such as acetyl (—C(═O)CH₃), propionyl(—C(═O)CH₂CH₃), isobutyryl ((—C(═O)CH(CH₃)₂), isovaleryl(—C(═O)CH₂CH(CH₃)₂), β-hydroxyisovaleryl (—C(═O)CH₂C(OH)(CH₃)₂),α-methyl-n-butyryl (—C(═O)CH(CH₃)CH₂CH₃), α,β-dimethylacryl((—C(═O)C(CH₃)═CHCH₃) and/or β,β-dimethylacryl ((—C(═O)CH═C(CH₃)₂).

Shikonin is an exemplary compound according to embodiments of theinvention (wherein R₃ and R₆ are OH, R₂, R₄ and R₅ are hydrogen, and R₁is —CH(OH)—CH═C(CH₃)₂).

In some of any of the respective embodiments described herein, R₁ is—CH₂CH₂CH═C(CH₃)₂, for example, in deoxyshikonin (wherein R₃ and R₆ areOH, and R₂, R₄ and R₅ are hydrogen).

Shikonin (or any other naphthoquinone compound described herein) mayalso be in a form of a glycoside of shikonin, for example, wherein oneor more OH of shikonin is replaced by a saccharide moiety (e.g., in aform of a substituted alkoxy group); and/or an ester of shikonin (or aglycoside of shikonin), for example, wherein one or more OH of shikonin(or a glycoside of shikonin) is replaced by a O-carboxy group (e.g.,—O—C(═O)CH₃).

The term “glycosyl”, as used herein, refers to a chemical group which isobtained by removing the hydroxyl group from the hemiacetal function ofa monosaccharide and, by extension, of a lower oligosaccharide (e.g., adisaccharide, a trisaccharide, etc.).

The term “glycoside”, as used herein, refers to a compound whichcomprises one or more glycosyl group (as defined herein). For example, a“shikonin glycoside” refers to shikonin attached to one or more glycosylgroup.

As used herein, the term “saccharide moiety” describes a moiety, asdefined herein, that contains one or more saccharide units.

As used herein the term “moiety” describes a major portion of a firstmolecule which is covalently linked to another molecule and whichretains its main structural features and/or activity. Thus, a “moiety”refers to a part of a molecule formed by conjugating the aforementionedfirst molecule to one or more other molecules, and represents thatportion of the first molecule that is present in the conjugationproduct. For example, a saccharide moiety may comprise all of asaccharide except for one hydroxyl group (e.g., as described hereinabovewith respect to glycosyl).

Accordingly, a “saccharide moiety” is that portion of a saccharidemolecule formed upon conjugating a second molecule (e.g., anaphthoquinone compound) thereto.

In exemplary embodiments of the invention, the saccharide moietycontains one saccharide unit and the saccharide unit is amonosaccharide.

The term “monosaccharide”, as used herein and is well known in the art,refers to a simple form of a sugar that consists of a single saccharideunit which cannot be further decomposed to smaller saccharide buildingblocks or moieties. Common examples of monosaccharides include glucose(dextrose), fructose, galactose, mannose, and ribose. Monosaccharidescan be classified according to the number of carbon atoms of thecarbohydrate, i.e., triose, having 3 carbon atoms such as glyceraldehydeand dihydroxyacetone; tetrose, having 4 carbon atoms such as erythrose,threose and erythrulose; pentose, having 5 carbon atoms such asarabinose, lyxose, ribose, xylose, ribulose and xylulose; hexose, having6 carbon atoms such as allose, altrose, galactose, glucose, gulose,idose, mannose, talose, fructose, psicose, sorbose and tagatose;heptose, having 7 carbon atoms such as mannoheptulose, sedoheptulose;octose, having 8 carbon atoms such as 2-keto-3-deoxy-manno-octonate;nonose, having 9 carbon atoms such as sialose; and decose, having 10carbon atoms. Monosaccharides are the building blocks ofoligosaccharides like sucrose (common sugar) and other polysaccharides(such as cellulose and starch).

The above monosaccharides encompass both D- and L-monosaccharides.

Alternatively, the monosaccharide can be a monosaccharide derivative, inwhich the saccharide unit comprises one or more substituents other thanhydroxyls. Such derivatives can be, but are not limited to, ethers,esters, amides, acids, phosphates and amines. Amine derivatives include,for example, glucosamine, galactosamine, fructosamine and mannosamine.Amide derivatives include, for example, N-acetylated amine derivativesof saccharides (e.g., N-acetylglucosamine, N-acetylgalactosamine).

The term “oligosaccharide” as used herein refers to a compound or moietythat comprises two or more linked monosaccharide units, as these aredefined herein. According to some embodiments of the present invention,an oligosaccharide comprises 2-6 monosaccharides. Alternatively, anoligosaccharide comprises 2-4 monosaccharides, or further alternatively,an oligosaccharide is a disaccharide moiety, having two monosaccharideunits.

The term “disaccharide” as used herein refers to a compound or moietythat comprises two linked monosaccharide units.

The term “trisaccharide” as used herein refers to a compound or moietythat comprises three linked monosaccharide units.

Non-limiting examples of shikonin glycosides (e.g.,shikonin-1′,8-di-O-β-D-glucopyranoside andshikonin-1′-O-β-D-glucopyranoside), and techniques which may optionallybe used to prepare them, are presented in Li et al. [Chem Pharm Bull(Tokyo) 2019, 67:1072-1075] and Su et al. [Eur J Med Chem 2010,45:2713-2718], each of which is incorporated herein by reference in itsentirety, especially with respect to the species of glycosides describedtherein.

A glycoside according to any of the respective embodiments describedherein may optionally comprise one or more (e.g., 1, 2 or 3)monosaccharide moieties (e.g., a glucose moiety, a galactose moiety,and/or a rhamnose moiety), one or more disaccharide moieties (e.g., aglucosyl-(1→2)-glucosyl and/or a rutinose moiety), and/or one or moretrisaccharide moieties (e.g., a glucosyl-(1→2)-glucosyl-(1→2)-glucosyland/or a 2^(G)-rhamnosylrutinose moiety).

The compounds and structures described herein encompass anystereoisomer, including enantiomers and diastereomers, of the compoundsdescribed herein, unless a particular stereoisomer is specificallyindicated.

Thus, for example, the term “shikonin” herein encompasses both the (R)and (S) enantiomers of5,8-dihydroxy-2-(1-hydroxy-4-methylpent-3-en-1-yl)naphthalene-1,4-dione,unless a specific enantiomer is indicated. For example, the term“alkannin” refers herein specifically to the (S) enantiomer.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems. In the context of the presentembodiments, a compound may exhibit one or more chiral centers, each ofwhich exhibiting an (R) or an (S) configuration and any combination, andcompounds according to some embodiments of the present invention, canhave any their chiral centers exhibit an (R) or an (S) configuration.

The term “diastereomers”, as used herein, refers to stereoisomers thatare not enantiomers to one another. Diastereomerism occurs when two ormore stereoisomers of a compound have different configurations at one ormore, but not all of the equivalent (related) stereocenters and are notmirror images of each other. When two diastereoisomers differ from eachother at only one stereocenter they are epimers. Each stereo-center(chiral center) gives rise to two different configurations and thus totwo different stereoisomers. In the context of the present invention,embodiments of the present invention encompass compounds with multiplechiral centers that occur in any combination of stereo-configuration,namely any diastereomer.

Imines and hydrazones of naphthoquinone compounds described herein,e.g., wherein one or more of the oxo (═O) groups of a compound ofFormula I is replaced by an ═N—R′ (imine) or ═N—NR′R″ (hydrazone),wherein R′ and R″ are as described herein, are encompassed by theinvention. Examples of such compounds include hydrazones formed from5-hydroxyquinone and hydrazine carboxamide or3,5-dinitrophenylhydrazine, as depicted in FIG. 1B.

In some of any of the respective embodiments, R₃ or R₄ or R₅ or R₆ is anaphthoquinone moiety which is also represented by Formula I, forexample, such that the compound may be regarded as a dimer of a compoundwherein R₃ or R₄ or R₅ or R₆ is hydrogen (or, if the aforementionednaphthoquinone moiety is likewise attached another naphthoquinonemoiety, as a trimer or longer oligomer of such a compound). In some suchembodiments, R₃ and R₆ are hydroxy, and R₄ or R₅ is the aforementionednaphthoquinone moiety.

Oligomerization of shikonin (including alkannin) is discussed in moredetail in Assimopoulou et al. [Biomed Chromatogr 2009, 23:182-198], thecontents of which are incorporated herein by reference (especially withrespect to the alkannin/shikonin-derived compounds described therein).

In some such embodiments, the naphthoquinone moiety is a substitutedaryl, e.g., such that compound comprises two naphthoquinones accordingto Formula I which are attached to one another, wherein eachnaphthoquinones may be regarded as an R₄ or R₅ substituent of the other.An example of such a compound is presented below (in which either of thetwo naphthoquinone moieties may be regarded as an R₄ substituent of theother naphthoquinone moiety).

In some such embodiments, the naphthoquinone moiety is a substitutedalkyl, e.g., an alkyl corresponding to R₁ according to any of therespective embodiments described herein, wherein the alkyl issubstituted by a naphthoquinone moiety. In some such embodiments, ahydroxy group comprised by the R₁ (according to any of the respectiveembodiments described herein) is replaced by a covalent bond attachingthe two naphthoquinone moieties. An example of such a compound ispresented below (in which the naphthoquinone moiety on the leftrepresents an R₄ substituent of the naphthoquinone moiety on the right).

As will be readily apparent to the skilled person, trimers and longeroligomers may be formed by essentially the same types of linkageexemplified in the dimers hereinabove, and/or using naphthoquinonecompounds other than shikonin as monomeric units (e.g., any compoundaccording to Formula I may optionally serve as a monomeric unit).

In some of any of the respective embodiments, the composition comprisesa compound having a molecular weight of about 420 to about 260 Da and/ora compound having a molecular weight of about 810 to about 850 Da. Insome such embodiments, the aforementioned compound(s) is a shikoninderivative, e.g., shikonin substituted by a saccharide moiety (e.g., amonosaccharide moiety which results in a molecular weight of about 453Da, or a trisaccharide moiety which results in a molecular weight ofabout 828 Da) or a shikonin trimer.

Exemplary naphthoquinone compounds include, without limitation,plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), shikonin, acetylshikonin, propionyl shikonin, isobutyryl shikonin, isovaleryl shikonin,β-hydroxyisovaleryl shikonin, α-methyl-n-butyryl shikonin,α,β-dimethylacryl shikonin, β,β-dimethylacryl shikonin,shikonin-1′-O-galactopyranoside, shikonin-1′-O-rhamnopyranoside,shikonin-1′-O-glucopyranoside, shikonin-1′,8-di-O-glucopyranoside,1′,5,8-tri-O-glucopyranoside,shikonin-1′-O-glucosyl-(1→2)-glucopyranoside,shikonin-1′-O-glucosyl-(1→2)-glucosyl-(1→2)-glucopyrano side,shikonin-1′-O-rutinoside, shikonin-1′-O-2^(G)-rhamnosylrutinoside, andany dimer or trimer of shikonin described in Assimopoulou et al. [BiomedChromatogr 2009, 23:182-198], including all stereoisomers thereof.

Further examples of naphthoquinone compounds include, withoutlimitation:

a) compounds of Formula I, in which R₃ and R₆ are each OH and R₂, R₄ andR₅ are each hydrogen, and:

R₁ is —CH═C(CH₃)₂;

R₁ is —CH₂CH═C(CH₃)₂;

R₁ is —C(═O)CH₂C═C(CH₃)₂;

R₁ is —C(═NOH)CH₂C═C(CH₃)₂;

R₁ is —C(═N—NH₂)CH₂C═C(CH₃)₂;

R₁ is —C(═NH)CH₂C═C(CH₃)₂;

R₁ is —CH(CO₂R)CH₂C═C(CH₃)₂ (wherein R is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl);

R₁ is —CH(OH)CH₂CH₂CH(CH₃)₂;

R₁ is —CH(OH)CH₂CH(OH)C(CH₃)₂OH;

R₁ is —CH(OH)CH₂CH(OH)CH(CH₃)₂;

R₁ is —CH(OH)CH₂C(═O)CH(CH₃)₂;

R₁ is —CH(OH)CH₂CH₂C(CH₃)₂OH;

R₁ is —CH(OH)CH₂C(═O)H;

R₁ is —CH(OH)CH₂C(═O)OH;

R₁ is —CH(OH)CH₂CH═CH—Ar (wherein Ar is aryl or heteroaryl);

R₁ is —CH(OH)CH₂CH═CR′R″ (wherein R′ and R″ are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl);

R₁ is —NHAr (wherein Ar is aryl or heteroaryl);

R₁ is —NHR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,or heteroalicyclic);

R₁ is —NHCH₂Ar (wherein Ar is aryl or heteroaryl);

R₁ is —N(R′)C(═O)OR″ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl);

R₁ is —N(R′)C(═O)R″ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl);

R₁ is —N(R′)C(═O)NR″R′″ (wherein R′, R″ and R′″ are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl);

R₁ is —SR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,or heteroalicyclic);

R₁ is —SAr (wherein Ar is aryl or heteroaryl);

R₁ is —SCH₂Ar (wherein Ar is aryl or heteroaryl);

R₁ is —S(═O)R (wherein R is hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalicyclic, aryl, or heteroaryl);

R₁ is —S(═O)₂R (wherein R is hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalicyclic, aryl, or heteroaryl);

R₁ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic,aryl, or heteroaryl;

R₁ is —CH(—OC(═O)NH₂)CH₂CH═CH(CH₃)₂;

R₁ is —CH(—OC(═O)NHC(═O)CH₃)CH₂CH═CH(CH₃)₂;

R₁ is —CH(—OC(═O)NHC(═O)CH₂Cl)CH₂CH═CH(CH₃)₂;

R₁ is —CH(—OC(═O)R)CH₂CH═CH(CH₃)₂ (wherein R is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, or heteroaryl);

R₁ is —CH(—OC(═O)NHR)CH₂CH═CH(CH₃)₂ (wherein R is hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, or heteroalicyclic);

R₁ is —CH(—OC(═O)NHAr)CH₂CH═CH(CH₃)₂ (wherein Ar is aryl or heteroaryl);

R₁ is —O—Ar (wherein Ar is aryl or heteroaryl);

R₁ is —OR (wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,or heteroalicyclic);

R₁ is —OCH₂Ar (wherein Ar is aryl or heteroaryl);

R₁ is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl),

b) compounds of Formula I, in which R₁ is —CH(OH)CH₂CH═C(CH₃)₂, R₃ andR₆ are each OH, and:

R₂ is halo or hydroxy, and R₄ and R₅ are each hydrogen;

R₄ is halo or hydroxy, and R₂ and R₅ are each hydrogen; and

R₅ is halo or hydroxy, and R₂ and R₄ are each hydrogen,

c) compounds of Formula I, in which R₃ and R₆ are each alkoxy, and R₂,R₄ and R₅ are each hydrogen, and:

R₁ is —CH(OR′)R″ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl);

R₁ is —CH(OH)R (wherein R is hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalicyclic, aryl, or heteroaryl); and

R₁ is —CHR′—OC(═O)OR″ (wherein R′ and R″ are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl, provided that R″ is not hydrogen),

d) compounds of Formula I, in which R₃ and R₆ are each alkoxy, R₁, R₂and R₄ are each hydrogen, and:

R₅ is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl); and

R₅ is —CH(OH)CH₂CH₂CH(CH₃)₂,

e) compounds of Formula I, in which R₃ and R₆ are each alkoxy, R₂, R₄and R₅ are each hydrogen, and:

R₁ is —CH(OR″)R′ (wherein R′ and R″ are each independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl); and

R₁ is —CHR′—OC(═O)OR″ (wherein R′ and R″ are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, orheteroaryl, with the proviso that R″ is not hydrogen),

f) compounds of Formula I, in which R₂ is methyl, R₃-R₆ are eachhydrogen, and R₁ is:—CH₂CH═C(CH₃)CH₂CH₂CH₂CH(CH₃)CH₂CH₂CH₂CH(CH₃)CH₂CH₂CH₂CH(CH₃)₂; or—CH₂CH═C(CH₃)CH₂CH₂CH═C(CH₃)CH₂CH₂CH═C(CH₃)CH₂CH₂CH═C(CH₃)₂, and

g) compounds depicted in FIG. 1A or FIG. 1B.

Without being bound by any particular theory, it is believed that manycommercially available preparations of shikonin comprise shikoninderivatives having one or more compound having a molecular weight asdescribed herein. It is further believed that administration of suchderivatives may provide a therapeutic effect similar to or even greaterthan shikonin per se (optionally by functioning as a prodrug of shikonin(e.g., decomposing in the body to form shikonin and a free saccharide),via enhancement of water solubility of shikonin by saccharide moieties,and or by providing a better fit to the active site with the active siteof 3CL protease (e.g., wherein each of the three hydroxy groups ofshikonin is substituted by a saccharide).

Shikonin and related compounds (according to any of the respectiveembodiments described herein) may optionally be derived from a plant ofthe borage family (Boraginaceae), optionally of the subfamilyBoraginoideae, and optionally of the tribe Lithospermeae.

Examples of borage family species which may optionally be used assources of shikonin or a related compound according to embodiments ofthe invention (according to any of the respective embodiments describedherein) include, without limitation, species of the genera Lithospermum(e.g., Lithospermum erythrorhizon), Lithidora, Onosma (e.g., Onosmavisianii, Onosma heterophylla, Onosma panicolatum, Onosma echioides),Echium (e.g., Echium plantagineum), Arnebia (e.g., Arnebia hispidissima,Arnebia euchroma, Arnebia tibetana, Arnebia guttata), and Alkanna (e.g.,Alkanna tinctoria).

The naphthoquinone compound (e.g., shikonin or a derivative thereof)according to any of the respective embodiment may optionally be producedusing plant tissue cultures (e.g., hairy root cultures), for example,using procedures such as described by Chaudhury & Pal [J Crop SciBiotech 2010 13:99-106] and/or Sim & Chang [Biotechnol Lett 1993,15:145-150].

The naphthoquinone compound (e.g., shikonin or a derivative thereof)according to any of the embodiments described herein may optionally bein a form of a product (e.g., a plant extract) which comprises thecompound, e.g., along with additional components, including a widevariety of compounds found, extracted and/or isolated from a plant.

For example, the naphthoquinone compound (e.g., shikonin or a derivativethereof) according to any of the embodiments described herein mayoptionally be part of a plant extract (e.g., an ethanol and/or waterextract), which may contain, for example, from about 0.5% to about 50%by weight of the naphthoquinone compound, and optionally from about 5%to about 40%, and optionally about 30% by weight. In some embodiments,at least a portion of the naphthoquinone compound (e.g., shikonin or aderivative thereof) in the plant extract is in a form of glycosideand/or a dimer or trimer thereof (according to any of the respectiveembodiments described herein). Shikonin and/or derivatives thereof mayoptionally be in a form of a composition (e.g., plant extract) describedas “zicao”, “zi cao”, “ying zi cao”, Arnebia or a variant thereof (e.g.,“Radix Arnebia” or “Arnebia euchrorna”), “purple gromwell”, “redgromwell”, “jichi”, “murasaki”, or variants thereof; and alkannin and/orderivatives thereof may optionally be in a form of a compositiondescribed as “alkanet extract” or “C. I. Natural Red 20”.

The naphthoquinone compound (e.g., shikonin or a derivative thereof)according to any of the embodiments described herein may optionally beprovided as a relatively pure preparation (e.g., at least 90% purity, orat least 95% purity). Such a relatively pure preparation may optionallybe combined with a composition with a lower concentration of the activecompound (e.g., a plant extract according to any of the respectiveembodiments described herein) in order to enhance the concentration ofactive compound in the plant extract (e.g., when a plant extract isconsiderably less costly than a relatively pure preparation).

In some of any of the embodiments described herein relating to ashikonin derivative, the shikonin derivative is a naphthoquinonecompound found, extracted and/or isolated from a plant source, e.g., aplant source according to any of the respective embodiments describedherein.

The naphthoquinone compound (e.g., shikonin or a derivative thereof)according to any of the embodiments described herein may also beprepared synthetically, using procedures known in the chemical arts.

Indications and Uses:

The compound of Formula I according to any of the respective embodimentsdescribed herein (e.g., in the respective section hereinabove) isoptionally for use in the treatment of a viral infection, for example, acoronavirus infection.

According to an aspect of the embodiments of the invention, there isprovided a use of a compound of Formula I according to any of therespective embodiments described herein (e.g., in the respective sectionhereinabove) in the manufacture of a medicament for use in the treatmentof a viral infection, for example, a coronavirus infection.

According to an aspect of the embodiments of the invention, there isprovided a method of treating a viral infection (for example, acoronavirus infection) in a (human or non-human) subject in needthereof, the method comprising administering to the subject a compoundof Formula I according to any of the respective embodiments describedherein (e.g., in the respective section hereinabove).

In some of any of the embodiments relating to treatment (according toany of the aspects described herein), the treatment is of a subject inwhich inhibiting inflammation would be beneficial, for example, in asubject considered to be afflicted by an excessive inflammatoryresponse, which is deleterious, e.g., to the quality of life and/orchance of survival of the subject.

In some of any of the embodiments relating to treatment, the treatmentcomprises administration of the compound (according to any of therespective embodiments described herein) at least once per day, andoptionally at least twice per day. In some such embodiments,administration is from 2 to 6 times per day, optionally 2, 3 or 4 timesper day.

In some of any of the embodiments relating to treatment, the treatmentcomprises oral administration of the compound (according to any of therespective embodiments described herein), for example, wherein oraladministration of the compound is effected at least 3 times per day,optionally four times per day.

In some of any of the respective embodiments, oral administration is ata dose in a range of from 10 mg to 5 grams, optionally from 50 mg to 1.5gram, optionally from 150 mg to 500 mg, and optionally about 200 mg.

In some of any of the respective embodiments, oral administration is ata dosage in a range of from 2 mg per kg per day to 200 mg per kg perday, optionally in a range of from 5 mg per kg per day to 100 mg per kgper day, and optionally about 20 mg per kg per day.

In some of any of the embodiments relating to treatment, the treatmentcomprises intravenous administration of the compound (according to anyof the respective embodiments described herein), for example, at a dosein a range of from 0.04 mg to 400 mg, optionally from 0.4 mg to 40 mg,and optionally about 4 mg.

In some of any of the embodiments relating to treatment, differentdosages are used for different degrees of treatment urgency, forexample, wherein a relatively low dosage (e.g., comprisingadministration twice per day of a relatively low amount of the compound,such as in a plant extract) is used as a prophylactic (e.g., following aknown exposure to the virus, and/or during an epidemic in which futureexposure to a virus has a relatively high probability); an intermediatedosage (e.g., comprising administration 3 times per day, optionally of aplant extract) is used to treat mild infections; and a relatively highdosage (e.g., comprising administration of a relatively high amount ofthe compound) is used to treat moderate and severe infections, forexample, characterized by pneumonia.

In some of any of the embodiments relating to treatment, thenaphthoquinone compound exhibits an antiviral activity, for example, anability to inhibit proliferation of a virus in the subject's body. Insome such embodiments, the naphthoquinone compound inhibits a protease(e.g., 3CL protease, or a corresponding virus which is essential forviral reproduction) of the virus (e.g., as determined using an assay forprotease activity such as described in the Examples section herein). Insome embodiments, the naphthoquinone compound inhibits autophagy (e.g.,thereby inhibiting proliferation of a virus which utilizes autophagy toproliferate). In some embodiments, the naphthoquinone compound bothinhibits a protease (e.g., 3CL protease) of the virus and inhibitsautophagy.

Herein, the term “autophagy” refers to a natural process within cellswhereby intracellular components are engulfed by vesicles (referred toas “autophagosomes”) which can deliver the engulfed components tolysosomes to be degraded, or are directly engulfed by lysosomes.

In some of any of the embodiments relating to treatment, thenaphthoquinone compound exhibits an anti-inflammatory effect, forexample, an ability to reduce an inflammatory response in a subject'sbody (e.g., as determined by C-reactive protein levels, interleukin 17(IL-17) and/or interleukin 6 (IL-6) levels). The anti-inflammatoryeffect is not a result of the antiviral effect, that is, is notdependent on first reducing a viral load in a subject. However, ananti-inflammatory effect may optionally cause an antiviral effect, forexample, wherein the virus utilizes an inflammatory effect to facilitateproliferation (e.g., by escaping infected cells and/or entering cellsupon damage of cells by the immune system). In some embodiments, thenaphthoquinone compound exhibits an anti-inflammatory effect (accordingto any of the respective embodiments described herein), as well as anantiviral effect (according to any of the respective embodimentsdescribed herein), such as protease inhibition and/or autophagyinhibition.

Without being bound by any particular theory, it is believed that ananti-inflammatory effect is particularly useful in treating some viralinfections (e.g., COVID-19), wherein much of the danger to a subject isassociated with excessive inflammatory response, e.g., conditionsassociated with a cytokine storm. It is further believed that ananti-inflammatory effect and antiviral effect may act in synergy intreating a subject. For example, slower viral replication due to anantiviral effect may be effective at reducing a risk of cytokine stormeven if it is not sufficiently potent to eliminate the virus.

Successful treatment outcomes include, without limitation, reduction ininflammation (e.g., as indicated by C-reactive protein levels),reduction in D-dimer levels (e.g., as an indicator of a reduction inover-coagulation), decrease in time until cure (as indicated by anegative result in a test for infection, e.g., by RT-PCR assay),decrease of hospitalization time, time until clinical improvement (e.g.,as defined by a National Early Warning Score 2 (NEWS2) of ≤2 maintainedfor 24 hours), and/or the subject reporting an improvement in feeling(e.g., relative to placebo). Additional optional parameters forassessing sickness/health include, e.g., changes in blood pressure,heart rate, respiratory rate, saturation and/or body temperature; numberof deaths in a group; incidence of deterioration and need of mechanicalventilation; and/or incidence and/or duration of time on supplementaloxygen.

Successful prophylactic treatment outcomes include, without limitation,avoidance of infection (in an individual), reduction of infection rate(in a population), and reduction or elimination of symptoms in infectedindividuals (e.g., wherein infection is indicated by production ofantibodies against the virus).

In some of any of the embodiments described herein relating totreatment, the treatment comprises administering at least one additionalactive agent, in addition to administration of the naphthoquinonecompound (e.g., according to any of the respective embodiments describedherein). Administration of the additional active agent(s) may optionallybe concomitant with or prior to or subsequent to administration of thenaphthoquinone compound. In some embodiments, administration of theadditional active agent(s) is effected at least twice per day, or atleast three times per day, or at least four times per day, optionallyfour times per day.

The at least one additional active agent may be, for example, a vitamin,N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, anantipyretic agent, an antiviral agent, and/or a protease inhibitor.

The protease inhibitor may be, for example, any protease inhibitordescribed U.S. Patent Publication No. 2004/0198716, which isincorporated herein by reference, particularly a protease inhibitordescribed therein which is not a naphthoquinone compound as describedherein. 5-methoxychromone is an example of such a protease inhibitor.

Examples of suitable vitamins include, without limitation, vitamin D(e.g., vitamin D3) and vitamin C, optionally in a liposomal formulation.

Examples of suitable anticoagulants include, without limitation,rivaroxaban, nafamostat, omega 3 fatty acids (and lipids comprisingthem), heparin and derivatives thereof (e.g., enoxaparin sodium orfondaparinux), epoprostenol, clopidogrel, argatroban and curcumin.

Examples of suitable anti-inflammatory agents include, withoutlimitation, ABX464; apremilast; atlizumab; baricitinib; berberine;cannabinoids, such as cannabidiol; celastrol; colchicine; curcuminoids,such as curcumin; decitabine; deferoxamine; DNase, such as dornase alfa;duvelisib; estradiol; elastase inhibitors, such as N-acetyl cysteine(NAC), freselestat, sivelestat, and/or any other compound depicted inFIG. 3 ; flavonoids (including substituted flavonoids, such asglycosides), such as quercetin and glycosides thereof (e.g., quercitrin,hyperoside, isoquercitrin and/or rutin), deoxykaempferol and glycosidesthereof, and/or epigallocatechin gallate; infliximab; opioids, such astramadol; palmitoylethanolamide (PEA); plant extracts, such as Boswelliaextract and henna (Lawsonia inermis) extract; polyphenols, such asellagic acid; stilbenoids, such as resveratrol and/orO-trimethyl-resveratrol); VB-201; NSAIDs (non-steroidalanti-inflammatory agents) such as aspirin, salicylate, salsalate,diflunisal, ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen,oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, diclofenac,aceclofenac, tolmetin, ketorolac, nabumetone, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam mefenamic acid, meclofenamicacid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib,valdecoxib, parecoxib, lumiracoxib, etoricoxib, nimesulide, niflumicacid, licofenac, and/or clonixin; and/or glucocorticoids, such asalclometasone and prodrugs thereof (e.g., alclometasone dipropionate),amcinonide, beclometasone and esters thereof (e.g., beclometasonedipropionate), betamethasone, budesonide, ciclesonide, chloroprednisoneand esters thereof (e.g., chloroprednisone 21-acetate), clobetasol andesters thereof (e.g., clobetasol propionate), clobetasone, clocortoloneand esters thereof (e.g., clocortolone pivalate), cloprednol, cortisol(a.k.a. hydrocortisone), cortisone, cortivazol, deflazacort, desonide,desoximetasone, dexamethasone, diflucortolone and esters thereof (e.g.,diflucortolone valerate), diflorasone and esters thereof (e.g.,diflorasone diacetate), difluprednate, fluclorolone and prodrugs thereof(e.g., fluclorolone acetonide), fludroxycortide, flumetasone and estersthereof (e.g., flumetasone pivalate), flunisolide, fluocinolone andprodrugs thereof (e.g., fluocinolone acetonide), fluocinonide,fluocortin, fluocortolone, fluperolone and esters thereof (e.g.,fluperolone acetate), fluprednidene and esters thereof (e.g.,fluprednidene acetate), fluticasone and esters thereof (e.g.,flutocasone furoate and fluticasone propionate), formocortal,halometasone, halcinonide, loteprednol, meprednisone,methylprednisolone, mometasone and esters thereof (e.g., mometasonefuroate), paramethasone, prednisolone, prednisone, prednylidene,rimexolone, RU-28362, tixocortol and esters thereof (e.g., tixocortolpivalate), triamcinolone and prodrugs thereof (e.g., triamcinoloneacetonide), and/or ulobetasol and esters thereof (e.g., ulobetasolpropionate).

Examples of suitable antipyretic agents include, without limitation,NSAIDs (such as listed herein), paracetamol, and dipyrone.

Examples of suitable antiviral agents (e.g., 3CLpro inhibitors) include,without limitation, bamlanivimab, casirivimab, chloroquine, clevudine,coronavir, DIFF-1 (2-hexanoyl-4,6-dichloro-5-methoxyresorcinol),favipiravir, ivermectin, GC376, hydroxychloroquine, imdevimab,lopinavir, nalidixic acid, nitazoxanide, remdesivir, ritonavir,rupintrivir, tenofovir and/or TMC-310911.

Additionally, the additional active agent may optionally be any agentdescribed for use in a clinical trial at the websitewww(dot)clinicaltrials(dot)gov/ct2/results?cond=COVID-19 as downloadedon Feb. 14, 2021, which is incorporated herein by reference in itsentirety, especially with respect to agents for treating a coronavirusinfection.

In some of any of the respective embodiments, N-acetyl cysteine isadministered at a dosage in a range of from 0.2 to 6.4 grams per day,and optionally from 0.8 to 1.6 grams per day.

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with a vitamin (e.g., vitamin D or vitamin C, as describedherein). In some such embodiments, the naphthoquinone compound isfurther used with N-acetyl cysteine, an anticoagulant, ananti-inflammatory agent, an antipyretic agent, an antiviral agent,and/or a protease inhibitor (e.g., according to any of the respectiveembodiments described herein).

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with an anticoagulant (e.g., an anticoagulant according toany of the respective embodiments described herein). In some suchembodiments, the naphthoquinone compound is further used with a vitamin,N-acetyl cysteine, an anti-inflammatory agent, an antipyretic agent, anantiviral agent, and/or a protease inhibitor (e.g., according to any ofthe respective embodiments described herein).

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with an anti-inflammatory agent (e.g., an anti-inflammatoryagent according to any of the respective embodiments described herein).In some such embodiments, the naphthoquinone compound is further usedwith a vitamin, N-acetyl cysteine, an anticoagulant, an antipyreticagent, an antiviral agent, and/or a protease inhibitor (e.g., accordingto any of the respective embodiments described herein).

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with an antipyretic agent (e.g., an antipyretic agentaccording to any of the respective embodiments described herein). Insome such embodiments, the naphthoquinone compound is further used witha vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatoryagent, an antiviral agent, and/or a protease inhibitor (e.g., accordingto any of the respective embodiments described herein).

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with an antiviral agent (e.g., an antiviral agent accordingto any of the respective embodiments described herein). In some suchembodiments, the naphthoquinone compound is further used with a vitamin,N-acetyl cysteine, an anticoagulant, an anti-inflammatory agent, anantipyretic agent, and/or a protease inhibitor (e.g., according to anyof the respective embodiments described herein).

According to some of any of the embodiments described herein for any ofthe compositions, methods and uses described herein, the naphthoquinonecompound as defined herein (e.g., a compound of Formula I) is used incombination with a protease inhibitor (e.g., a protease inhibitoraccording to any of the respective embodiments described herein). Insome such embodiments, the naphthoquinone compound is further used witha vitamin, N-acetyl cysteine, an anticoagulant, an anti-inflammatoryagent, an antipyretic agent, and/or an antiviral agent (e.g., accordingto any of the respective embodiments described herein).

According to an aspect of some embodiments, there is provided a methodof inhibiting a coronavirus 3CL protease, the method comprisingcontacting the 3CL protease with a compound of Formula I according toany of the respective embodiments described herein (e.g., in therespective section hereinabove).

The method of inhibiting a coronavirus 3CL protease may optionally beeffected in vitro and/or in vivo, in human or non-human subject (e.g., amammal). In some embodiments, the method comprises administering thecompound (according to any of the respective embodiments describedherein) to a subject in need thereof (e.g., according to any of theembodiments described herein relating to subjects, treatment regimensand/or indications).

When the 3CL activity is inhibited in a subject, the method according tothis aspect optionally further comprises inhibiting inflammation and/orautophagy in the subject (according to any of the respective embodimentsdescribed herein).

According to an aspect of some embodiments, there is provided a methodof treating a coronavirus infection in a subject in need thereof, themethod comprising administering to the subject at least one compoundthat exhibits at least two of (and optionally each of) the followingproperties:

-   -   (i) inhibition of an activity of a 3CL protease of the        coronavirus;    -   (ii) inhibition of inflammation in the subject (optionally        comprising reduction and/or prevention of a cytokine storm by        inhibition of a kinase, such as Janus kinase (JAK); and    -   (iii) inhibition of autophagy in the subject.

The at least one compound may optionally comprise a single compoundwhich exhibits the at least two properties described herein; or comprisea plurality of compounds which exhibits the at least two propertiesdescribed herein; and/or comprise compounds which exhibit differentproperties, e.g., such that the at least two properties are exhibited bythe combination of compounds but not by an individual compound.

The at least one compound according to this aspect optionally, but notnecessarily, comprises a compound according to Formula I according toany of the respective embodiments described herein. In some embodiments,the treatment comprises administering, in addition to the compoundaccording to Formula I, at least one additional agent that exhibits theabovementioned inhibition of activity of 3CL protease, inhibition ofinflammation, and/or inhibition of autophagy, optionally supplementingone or two inhibitory activities which in the compound of Formula I iseither weak or absent.

In some embodiments, the treatment comprises administering, in additionto the compound according to Formula I, at least one additional agentthat exhibits the abovementioned inhibition of activity of 3CL protease,and optionally also inhibition of inflammation, and/or inhibition ofautophagy.

In some embodiments, the treatment comprises administering, in additionto the compound according to Formula I, at least one additional agentthat exhibits the abovementioned inhibition of inflammation, andoptionally also inhibition of activity of 3CL protease and/or inhibitionof autophagy.

In some embodiments, the treatment comprises administering, in additionto the compound according to Formula I, at least one additional agentthat exhibits the abovementioned inhibition of autophagy, and optionallyalso inhibition of activity of 3CL protease and/or inhibition ofinflammation.

The coronavirus according to any of the respective embodiments describedherein is optionally a betacoronavirus, for example, an embecovirus(a.k.a. lineage A), sarbecovirus (a.k.a. lineage B), merbecovirus(a.k.a. lineage C), nobecovirus (a.k.a. lineage D), and hibecovirus.Exemplary betacoronaviruses include SARS-related coronavirus (a speciesof sarbecovirus), human coronavirus OC43, and human coronavirus HKU1,including any strains thereof (e.g., SARS-CoV-2).

Alternatively or additionally, the coronavirus may optionally beassociated with the common cold, such as 229E, NL63, HKU1 and OC43coronaviruses.

In some embodiments, the viral infection treatable using anaphthoquinone compound described herein is associated with a virusother than a coronavirus, for example, CMV (cytomegalovirus), HRV (humanrhinoviruses), hepatovirus A, HMV (human meningo virus), and/or HIV(human immunodeficiency virus).

Pharmaceutical Compositions:

For any use and/or indication described herein, the naphthoquinonecompound and/or additional active agent(s) of some embodiments of theinvention can be administered to an organism per se, or in a form of apharmaceutical composition, which may optionally further comprisesuitable carriers or excipients.

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to one or more agentaccountable for the biological effect(s), for example, thenaphthoquinone compound and/or additional active agent(s) according toany of the respective embodiments described herein. In some of any ofthe embodiments relating to a composition, the composition comprises oneor more naphthoquinone compound (according to any of the respectiveembodiments described herein) and one or more additional active agent(according to any of the respective embodiments described herein).

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

In some embodiments of any of the embodiments described herein, thenaphthoquinone compound and/or additional active agent(s) (according toany of the respective embodiments described herein), are co-formulatedin a single pharmaceutical composition.

Alternatively, in some embodiments of any of the embodiments describedherein, each of the naphthoquinone compound and/or additional activeagent(s) (according to any of the respective embodiments describedherein) is formulated individually in a pharmaceutical composition. Thecomposition comprising the naphthoquinone compound may optionally beco-administered with one or more composition comprising an additionalactive agent(s).

The naphthoquinone compound may be present in a composition atessentially any concentration, for example, from 0.01% to 99.99% byweight, optionally, from 0.1% to 50% by weight.

In some embodiments of any of the respective embodiments describedherein, the composition comprising the naphthoquinone compound furthercomprises phospholipids. The weight ratio of phospholipids to thenaphthoquinone compound in the composition is optionally in a range offrom 10:1 to 1:10, and optionally from 3:1 to 1:3. In some exemplaryembodiments, the phospholipid to naphthoquinone weight ratio is about1:1.

The phospholipids (according to any of the respective embodimentsdescribed herein) are optionally composed primarily ofphosphatidylcholine, that is, at least 50% (by weight) of thephospholipids are phosphatidylcholine. Optionally, at least 60% or atleast 70% or at least 80% or at least 90% of the phospholipids arephosphatidylcholine (by weight).

The phospholipids (according to any of the respective embodimentsdescribed herein) optionally comprise phosphatidylserine, for example,such that at least 10% or at least 20% by weight of the phospholipids isphosphatidylserine. In some exemplary embodiments, the proportion of thephosphatidylserine in the phospholipids is about 20% by weight. In someof any of the aforementioned embodiments, the phospholipids areoptionally composed primarily of phosphatidylcholine (according to anyof the respective embodiments described herein).

Without being bound by any particular theory, it is believed thatphospholipids (e.g., in an amount described herein) facilitateabsorption of active ingredients into the blood (e.g., upon oraladministration).

In some embodiments of any of the respective embodiments describedherein, the composition further comprises liposomes, which optionallyenvelop at least a portion of the active ingredient(s). The liposomesmay optionally comprise phospholipids (e.g., according to any of therespective embodiments described herein), for example, in combinationwith an aqueous carrier.

Alternatively, the composition may optionally be a dry composition,which forms liposomes upon contact with water.

Techniques for formulation and administration of drugs (according to anyof the aspects of embodiments of the invention described herein) may befound in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

In some embodiments of any of the embodiments described herein,administration of a naphthoquinone compound and/or additional activeagent(s) is systemic.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodimentsof the invention thus may be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups (e.g., foradministration to children, optionally as a prophylactic treatment forhealthy individuals), slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification, or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to some embodiments of the invention are convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodiumcarboxymethylcellulose, sorbitol or dextran. Optionally, the suspensionmay also contain suitable stabilizers or agents that increase thesolubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of someembodiments of the invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount means anamount of active ingredients (naphthoquinone compound and/or additionalactive agent(s) described herein) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., coronavirus infection) orprolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to providelevels of the active ingredient that are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed herein.

Additional Definitions:

As used herein throughout, the term “alkyl” refers to any saturatedaliphatic hydrocarbon including straight chain and branched chaingroups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever anumerical range; e.g., “1 to 20”, is stated herein, it implies that thegroup, in this case the hydrocarbon, may contain 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Morepreferably, the alkyl is a medium size alkyl having 1 to 10 carbonatoms. Most preferably, unless otherwise indicated, the alkyl is a loweralkyl having 1 to 4 carbon atoms. The alkyl group may be substituted ornon-substituted. When substituted, the substituent group can be, forexample, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl,sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl,phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, as these terms are defined herein.

Herein, the term “alkenyl” describes an unsaturated aliphatichydrocarbon comprise at least one carbon-carbon double bond, includingstraight chain and branched chain groups. Preferably, the alkenyl grouphas 2 to 20 carbon atoms. More preferably, the alkenyl is a medium sizealkenyl having 2 to 10 carbon atoms. Most preferably, unless otherwiseindicated, the alkenyl is a lower alkenyl having 2 to 6 carbon atoms.The alkenyl group may be substituted or non-substituted. Substitutedalkenyl may have one or more substituents, whereby each substituentgroup can independently be, for example, alkynyl, cycloalkyl, alkynyl,aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate,sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime,hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl,hydrazine, hydrazide, thiohydrazide, and amino.

Herein, the term “alkynyl” describes an unsaturated aliphatichydrocarbon comprise at least one carbon-carbon triple bond, includingstraight chain and branched chain groups. Preferably, the alkynyl grouphas 2 to 20 carbon atoms. More preferably, the alkynyl is a medium sizealkynyl having 2 to 10 carbon atoms. Most preferably, unless otherwiseindicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms.The alkynyl group may be substituted or non-substituted. Substitutedalkynyl may have one or more substituents, whereby each substituentgroup can independently be, for example, cycloalkyl, alkenyl, aryl,heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate,sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime,hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl,hydrazine, hydrazide, thiohydrazide, and amino.

A “cycloalkyl” group refers to a saturated on unsaturated all-carbonmonocyclic or fused ring (i.e., rings which share an adjacent pair ofcarbon atoms) group wherein one of more of the rings does not have acompletely conjugated pi-electron system. Examples, without limitation,of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane,cyclopentene, cyclohexane, cyclohexadiene, cycloheptane,cycloheptatriene, and adamantane. A cycloalkyl group may be substitutedor non-substituted. When substituted, the substituent group can be, forexample, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone,carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl,hydrazine, hydrazide, thiohydrazide, and amino, as these terms aredefined herein. When a cycloalkyl group is unsaturated, it may compriseat least one carbon-carbon double bond and/or at least one carbon-carbontriple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)having a completely conjugated pi-electron system. Examples, withoutlimitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. Thearyl group may be substituted or non-substituted. When substituted, thesubstituent group can be, for example, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl,sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo,imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, athiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido,guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, asthese terms are defined herein.

A “heteroaryl” group refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) having in the ring(s) one or moreatoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or non-substituted. When substituted, the substituent groupcan be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate,sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime,hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl,hydrazine, hydrazide, thiohydrazide, and amino, as these terms aredefined herein.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or non-substituted. When substituted,the substituted group can be, for example, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl,sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo,imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, athiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido,guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, asthese terms are defined herein. Representative examples are piperidine,piperazine, tetrahydrofuran, tetrahydropyran, morpholine and the like.

Herein, the terms “amine” and “amino” each refer to either a —NR′R″group or a —N⁺R′R″R′″ group, wherein R′, R″ and R′″ are each hydrogen ora substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl,heteroalicyclic (linked to amine nitrogen via a ring carbon thereof),aryl, or heteroaryl (linked to amine nitrogen via a ring carbonthereof), as defined herein. Optionally, R′, R″ and R′″ are hydrogen oralkyl comprising 1 to 4 carbon atoms. Optionally, R′ and R″ (and R′″, ifpresent) are hydrogen. When substituted, the carbon atom of an R′, R″ orR′″ hydrocarbon moiety which is bound to the nitrogen atom of the amineis not substituted by oxo (unless explicitly indicated otherwise), suchthat R′, R″ and R′″ are not (for example) carbonyl, C-carboxy or amide,as these groups are defined herein.

An “azide” group refers to a —N═N⁺═N⁻ group.

An “alkoxy” group refers to any of an —O-alkyl, —O-alkenyl, —O-alkynyl,—O-cycloalkyl, and —O-heteroalicyclic group, as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “hydroxy” group refers to a —OH group.

A “thiohydroxy” or “thiol” group refers to a —SH group.

A “thioalkoxy” group refers to any of an —S-alkyl, —S-alkenyl,—S-alkynyl, —S-cycloalkyl, and —S-heteroalicyclic group, as definedherein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “carbonyl” group refers to a —C(═O)—R′ group, where R′ is defined ashereinabove.

A “thiocarbonyl” group refers to a —C(═S)—R′ group, where R′ is asdefined herein.

A “C-carboxy” group refers to a —C(═O)—O—R′ group, where R′ is asdefined herein.

An “O-carboxy” group refers to an R′C(═O)—O— group, where R′ is asdefined herein.

A “carboxylic acid” group refers to a —C(═O)OH group.

An “oxo” group refers to a ═O group.

An “imine” group refers to a ═N—R′ group, where R′ is as defined herein.

An “oxime” group refers to a ═N—OH group.

A “hydrazone” group refers to a ═N—NR′R″ group, where each of R′ and R″is as defined herein.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “sulfinyl” group refers to an —S(═O)—R′ group, where R′ is as definedherein.

A “sulfonyl” group refers to an —S(═O)₂—R′ group, where R′ is as definedherein.

A “sulfonate” group refers to an —S(═O)₂—O—R′ group, where R′ is asdefined herein.

A “sulfate” group refers to an —O—S(═O)₂—O—R′ group, where R′ is asdefined as herein.

A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamidoand N-sulfonamido groups, as defined herein.

An “S-sulfonamido” group refers to a —S(═O)₂—NR′R″ group, with each ofR′ and R″ as defined herein.

An “N-sulfonamido” group refers to an R'S(═O)₂—NR″— group, where each ofR′ and R″ is as defined herein.

An “O-carbamyl” group refers to an —OC(═O)—NR′R″ group, where each of R′and R″ is as defined herein.

An “N-carbamyl” group refers to an R′OC(═O)—NR″— group, where each of R′and R″ is as defined herein.

An “O-thiocarbamyl” group refers to an —OC(═S)—NR′R″ group, where eachof R′ and R″ is as defined herein.

An “N-thiocarbamyl” group refers to an R′OC(═S)NR″— group, where each ofR′ and R″ is as defined herein.

An “S-thiocarbamyl” group refers to an —SC(═O)—NR′R″ group, where eachof R′ and R″ is as defined herein.

An “amide” or “amido” group encompasses C-amido and N-amido groups, asdefined herein.

A “C-amido” group refers to a —C(═O)—NR′R″ group, where each of R′ andR″ is as defined herein.

An “N-amido” group refers to an R′C(═O)—NR″— group, where each of R′ andR″ is as defined herein.

A “urea group” refers to an —N(R′)—C(═O)—NR″R′″ group, where each of R′,R″ and R″ is as defined herein.

A “thiourea group” refers to a —N(R′)—C(═S)—NR″R′″ group, where each ofR′, R″ and R″ is as defined herein.

A “nitro” group refers to an —NO₂ group.

A “cyano” group refers to a —C≡N group.

The term “phosphonyl” or “phosphonate” describes a —P(═O)(OR′)(OR″)group, with R′ and R″ as defined hereinabove.

The term “phosphate” describes an —O—P(═O)(OR′)(OR″) group, with each ofR′ and R″ as defined hereinabove.

The term “phosphinyl” describes a —PR′R″ group, with each of R′ and R″as defined hereinabove.

The term “hydrazine” describes a —NR′—NR″R′″ group, with R′, R″, and R′″as defined herein.

As used herein, the term “hydrazide” describes a —C(═O)—NR′—NR″R′″group, where R′, R″ and R′ are as defined herein.

As used herein, the term “thiohydrazide” describes a —C(═S)—NR′—NR″R′″group, where R′, R″ and R′″ are as defined herein.

A “guanidinyl” group refers to an —RaNC(═NRd)-NRbRc group, where each ofRa, Rb, Rc and Rd can be as defined herein for R′ and R″.

A “guanyl” or “guanine” group refers to an RaRbNC(═NRd)- group, whereRa, Rb and Rd are as defined herein.

For any of the embodiments described herein, the compound describedherein may be in a form of a salt, for example, a pharmaceuticallyacceptable salt, and/or in a form of a prodrug.

As used herein, the phrase “pharmaceutically acceptable salt” refers toa charged species of the parent compound and its counter-ion, which istypically used to modify the solubility characteristics of the parentcompound and/or to reduce any significant irritation to an organism bythe parent compound, while not abrogating the biological activity andproperties of the administered compound. A pharmaceutically acceptablesalt of a compound as described herein can alternatively be formedduring the synthesis of the compound, e.g., in the course of isolatingthe compound from a reaction mixture or re-crystallizing the compound.

In the context of some of the present embodiments, a pharmaceuticallyacceptable salt of the compounds described herein may optionally be anacid addition salt and/or a base addition salt.

An acid addition salt comprises at least one basic (e.g., amine and/orguanidinyl) group of the compound which is in a positively charged form(e.g., wherein the basic group is protonated), in combination with atleast one counter-ion, derived from the selected acid, that forms apharmaceutically acceptable salt. The acid addition salts of thecompounds described herein may therefore be complexes formed between oneor more basic groups of the compound and one or more equivalents of anacid.

A base addition salt comprises at least one acidic (e.g., carboxylicacid) group of the compound which is in a negatively charged form (e.g.,wherein the acidic group is deprotonated), in combination with at leastone counter-ion, derived from the selected base, that forms apharmaceutically acceptable salt. The base addition salts of thecompounds described herein may therefore be complexes formed between oneor more acidic groups of the compound and one or more equivalents of abase.

Depending on the stoichiometric proportions between the charged group(s)in the compound and the counter-ion in the salt, the acid additionssalts and/or base addition salts can be either mono-addition salts orpoly-addition salts.

The phrase “mono-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and charged formof the compound is 1:1, such that the addition salt includes one molarequivalent of the counter-ion per one molar equivalent of the compound.

The phrase “poly-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and the chargedform of the compound is greater than 1:1 and is, for example, 2:1, 3:1,4:1 and so on, such that the addition salt includes two or more molarequivalents of the counter-ion per one molar equivalent of the compound.

An example, without limitation, of a pharmaceutically acceptable saltwould be an ammonium cation or guanidinium cation and an acid additionsalt thereof, and/or a carboxylate anion and a base addition saltthereof.

The base addition salts may include a cation counter-ion such as sodium,potassium, ammonium, calcium, magnesium and the like, that forms apharmaceutically acceptable salt.

The acid addition salts may include a variety of organic and inorganicacids, such as, but not limited to, hydrochloric acid which affords ahydrochloric acid addition salt, hydrobromic acid which affords ahydrobromic acid addition salt, acetic acid which affords an acetic acidaddition salt, ascorbic acid which affords an ascorbic acid additionsalt, benzenesulfonic acid which affords a besylate addition salt,camphorsulfonic acid which affords a camphorsulfonic acid addition salt,citric acid which affords a citric acid addition salt, maleic acid whichaffords a maleic acid addition salt, malic acid which affords a malicacid addition salt, methanesulfonic acid which affords a methanesulfonicacid (mesylate) addition salt, naphthalenesulfonic acid which affords anaphthalenesulfonic acid addition salt, oxalic acid which affords anoxalic acid addition salt, phosphoric acid which affords a phosphoricacid addition salt, toluenesulfonic acid which affords ap-toluenesulfonic acid addition salt, succinic acid which affords asuccinic acid addition salt, sulfuric acid which affords a sulfuric acidaddition salt, tartaric acid which affords a tartaric acid addition saltand trifluoroacetic acid which affords a trifluoroacetic acid additionsalt. Each of these acid addition salts can be either a mono-additionsalt or a poly-addition salt, as these terms are defined herein.

As used herein, the term “prodrug” refers to a compound which isconverted in the body to an active compound (e.g., the compound of theformula described hereinabove). A prodrug is typically designed tofacilitate administration, e.g., by enhancing absorption. A prodrug maycomprise, for example, the active compound modified with ester groups,for example, wherein any one or more of the hydroxyl groups of acompound is modified by an acyl group, optionally (C₁₋₄)acyl (e.g.,acetyl) group to form an ester group, and/or any one or more of thecarboxylic acid groups of the compound is modified by an alkoxy oraryloxy group, optionally (C₁₋₄)alkoxy (e.g., methyl, ethyl) group toform an ester group.

Further, each of the compounds described herein, including the saltsthereof, can be in a form of a solvate or a hydrate thereof.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the heterocyclic compounds described herein) and a solvent,whereby the solvent does not interfere with the biological activity ofthe solute.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

The compounds described herein can be used as polymorphs and the presentembodiments further encompass any isomorph of the compounds and anycombination thereof.

As used herein the term “about” refers to ±20% or ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Example 1

As a general procedure, shikonin or a composition comprising shikonin ora derivative thereof is formulated in capsules, optionally incombination with lecithin (phospholipids, comprising primarilyphosphatidylcholine) (e.g., at a shikonin-to-lecithin weight ratio ofabout 1:1). The shikonin or derivative thereof may be substantially pure(from a synthetic or natural source) or a part of an extract of a plant,such as Lithospermum erythrorhizon, Arnebia euchroma or another memberof the borage family.

Using the above general procedure, an extract of purple gromwell(Lithospermum erythrorhizon) root (zicao) was prepared using anappropriate solvent, followed by spray drying and sieving, to obtain apurple powder. 175 mg of the powdered purple gromwell extract,containing about 30% shikonin and/or derivatives thereof, was placedwith an equal weight of lecithin (Lipoid® PS P 20×, obtained from LipoidGmbH) in Capsugel® delayed release (DR) capsules.

As an alternative to capsules, a syrup was prepared comprising lecithinand shikonin (95% purity) at a 5:1 lecithin:shikonin ratio, 44% alcoholas solvent, and honey.

Based on literature reports, toxicity of shikonin is not expected atdosages of less than 8 grams per day.

Example 2 Effect of Shikonin on 3CL Protease of SARS-Coronavirus 2 in anIn Vitro Assay

Inhibition of the activity of 3CL protease of SARS-coronavirus 2 (thecoronavirus associated with COVID-19) was determined by an assay basedon commercial kit (BPS cat. 79955-1). 150 ng of 3CL protease wasincubated with progressively smaller shikonin concentrations for 30minutes. Shikonin was obtained from NLC pharma and was 95% pure. Thereaction was initiated by addition of 50 μl of 50 μM kit substrate (25μM final concentration), and then fluorescence was monitored (at 340 nmexcitation wavelength, 510 nm emission wavelength) using a CLARIOstar®Plus plate reader (BMG Labtech). The 3CL protease activity wasquantified as relative fluorescent units (RFU) per minute.

As shown in Table 1, the concentration at which shikonin inhibited 3CLprotease by 50% (IC50) was about 10 and almost complete inhibition of3CL protease was exhibited at a concentration of 100 μM shikonin.

These results indicate that shikonin is capable of inhibitingSARS-coronavirus 2 3CL protease.

TABLE 1 Enzymatic activity of 3CL protease in presence of variousconcentrations of shikonin, as determined by fluorescent assay (mean ±standard deviation) Shikonin Activity concentration (RFU/minute) 100 μM 2.9 ± 18.4 10 μM 425.0 ± 7.8  1 μM 680.1 ± 34.3 0.1 μM 653.8 ± 28.1 0μM 743.7 ± 20.6

Example 3 Effect of Purple Gromwell Extract on Patients with COVID-19

The efficacy of an exemplary purple gromwell extract, prepared andformulated in capsules as described in Example 1, was evaluated in aclinical trial in patients afflicted by COVID-19. In one group, 10patients received 175 mg of purple gromwell extract and lecithin, 4-8times per day. In the other group, 19 patients received vitamin C andspirulina dietary supplement (control group). All patients were at anage in the range of 60-90 years.

In the group treated with purple gromwell extract, all 10 patientssurvived, 1 of the 10 (10%) needed artificial respiration, and theaverage time until release from the hospital was about 6-7 days. Incontrast, in the other group, 5 patients (26%) died, 5 (26%) neededartificial respiration, and the average time until release from thehospital was about 3 weeks.

As shown in FIG. 2 , patients treated with purple gromwell extractexhibited considerably lower levels of C-reactive protein (CRP) andferritin (both of which are proteins associated with inflammation) thandid control patients.

Indeed, in most cases, CRP levels of purple gromwell extract-treatedpatients normalized within several days, with typical levels of 4-20mg/liter upon leaving the hospital.

High D-dimer levels were observed in six patients. One of the six wastreated with purple gromwell extract and the D-dimer levels thenreturned to normal; whereas five received the control treatment, andnone of the five survived.

These results indicate that purple gromwell extract is effective attreating viral diseases such as COVID-19 and reducing inflammation in aclinical setting.

In addition, the following case studies are presented:

Case 1: A 74 year-old male, with a history of bronchial asthma and COPD(being treated daily, including with oxygen), hypertension and diabetesmellitus, was prophylactically administered capsules containing purplegromwell extract daily for 6 months during the COVID-19 pandemic (withno other antiviral treatment). The subject was very socially active, incontact with many people. Two of the subject's assistants and his driverdeveloped COVID-19 while working in his close presence, but the subjectexhibited no signs of infection (including normal CPR test results).

Case 2: A 60 year-old male, with a history of ischemic heart disease,hypertension, excess weight, and appropriate treatments (including bloodthinner injections), was diagnosed with COVID-19 (by PCR assay)following cough, weakness, and observation (by x-ray) of bilateralextended bronco-pneumonia. Following 24 hours of antibiotic and steroidtreatment, his condition deteriorated and administration of vitamins andcapsules containing purple gromwell extract began. About 48-72 hourslater, the situation began to improve, and four days later, theshortness of breath and cough almost disappeared.

Case 3: A 68 year-old male, with no relevant medical history, washospitalized with an oxygen saturation of 59%. COVID-19 and massivebilateral pneumonia were confirmed, and a treatment of antibiotics,steroids and blood thinner was initiated. His condition deteriorated(oxygen saturation of 10-20%) and he was intubated and put on anICU-ventilator. Upon ventilation for 48 hours, no improvement wasobserved. A syrup (prepared as described in Example 1) comprisingshikonin was then administered via a naso-gastric tube. At this stage,CRP levels were about 300. About 48-72 hours later, CRP levels droppedto 65, and a few days later reverted to normal. Shortly thereafter,oxygen saturation increased. The subject recovered and was released fromthe hospital 3 weeks later.

Case 4: An 81 year-old female, with a history of hypertension, diabetesmellitus and peripheral vascular disease (underwent braincatheterization), was diagnosed with COVID-19 (by PCR assay) afterexhibiting progressively worsening shortness of breath. Treatment athome with oxygenation and steroids was initiated. The subject thendeveloped fever and a cough, was diagnosed with pneumonia and began toreceive antibiotics. After four days, the subject's health deterioratedfurther, leading to hospitalization. Oxygen saturation was 85%, andtreatment with a respirator and remdesivir began. After five days, thecondition was worse, with CRP levels of 120. The subject was thenadministered purple gromwell extract capsules and then attached to anECMO unit. About 12 hours later, oxygen saturation stabilized at about98%, and the following morning CRP levels decreased to 65. Four dayslater, the CRP levels decreased further to 15 and then decreased tonormal; and clinical improvement was observed. The subject was releasedfrom the hospital 8-9 days after the initiation of the purple gromwellextract treatment.

Example 4 Preparation of Exemplary Capsule Formulation

RX-type capsules (capable of passing through the stomach and decomposingin the small intestines) are prepared with 200 mg of shikoninglycosides, as well as 200 mg of lecithin and a small amount ofmagnesium stearate (a lubricant). The shikonin glycosides comprise about40% by weight monoglucosyl shikonin and about 60% by weight triglucosylshikonin, and are relatively water-soluble.

Example 5 Preparation of Exemplary Capsule Formulation

Capsules are prepared with 250 mg of a root extract powder (prepared asdescribed in Example 1 or obtained from a commercial source) and 250 mglecithin with about 20% phosphatidylserine (Lipoid® PS P 20×, obtainedfrom Lipoid GmbH). The root extract powder optionally comprises about30% shikonin or derivative thereof by weight, such that the amount ofactive ingredient is about 75 mg.

Example 6 Preparation of Exemplary Capsule Formulation

Size 00 acid-resistant capsules (obtained from CapsCanada) are filledwith a mixture of:

135 mg microcrystalline cellulose (Avicel® PH102, obtained from DuPontNutrition),

100 mg lecithin with about 20% phosphatidylserine (Lipoid® PS P 20×,obtained from Lipoid GmbH),

48 mg polyethylene glycol (PEG 3000, obtained from Merck),

33 mg poloxamer 407 (Kolliphor® P407, obtained from BASF),

120 mg shikonin (84.7% potency, obtained from Henan Steeda IndustrialCo. Ltd.), and

100 mg purple gromwell root extract powder (obtained from Imaherb).

The microcrystalline cellulose, shikonin, poloxamer and polyethyleneglycol are ground, and then mixed with the lecithin and root extractpowder, in suitable ratios. For example, 3752 grams of uniform mixtureis used to fill 7000 capsules with 536 mg (±27 mg) of mixture percapsule.

The 120 mg shikonin of relatively high purity allows for a relativelyhigh dose of shikonin, which is more difficult to obtain using only rootextract powders. Such high-dose formulations are particularly suitable,for example, for treating moderate and severe cases of coronavirusinfection.

Example 7 Effect of Exemplary Formulation on Patients with COVID-19

The efficacy of an exemplary shikonin-containing composition is assessedin a randomized, double-blind, placebo-controlled clinical study inhospitalized COVID-19 patients with SARS-CoV-2 infection confirmed (byRT-PCR assay). The administered composition is a liposomal formulationof shikonin prepared as described in Example 6.

Patients under the weight of 60 kg are administered 2 capsules, 3 timesper day (6 capsules per day); patients within the weight range of 60-80kg are administered 2 capsules, 4 times per day (8 capsules per day);and patients over the weight of 80 kg are administered 3 capsules, 4times per day (12 capsules per day). Administration is during days 1-10of the patient's hospitalization. Placebo capsules (comprising the samecarrier without the active ingredients) are administered to an equallysized group of patients. Both groups receive standard of care forCOVID-19 in addition to the capsules.

The effect of the shikonin-containing composition on sickness severity(relative to placebo) is assessed. Severity of sickness is evaluated astime until hospital discharge and/or time until clinical improvement asdefined by a National Early Warning Score 2 (NEWS2) of ≤2 maintained for24 hours.

Additional parameters for assessing sickness/health include, e.g.,changes in blood pressure, heart rate, respiratory rate, saturationand/or body temperature; time from first day of treatment until negativetest result (by RT-PCR assay) for virus; number of deaths in group;incidence of deterioration and need of mechanical ventilation; and/orincidence and/or duration of time on supplemental oxygen.

Example 8 Effect of Arnebia euchroma Extract on 3CL Protease ofSARS-Coronavirus 2 in an In Vitro Assay

Inhibition of the activity of recombinant 3CL protease ofSARS-coronavirus 2 by Arnebia euchroma extract was determined. Theextract was identified by the manufacturer as containing 30% shikonin,but appeared upon HPLC examination to contain shikonin only in the formof glycosides, e.g., at a molecular weight of about 800 Da.

A fluorescence assay was performed using 96-well black non-binding flatbottom plates (Greiner Bio-One), with a reaction volume of 100 μl. 50 μlof 150 ng 3CL protease was incubated with the extract in reaction bufferfor 30 minutes at room temperature. A reaction was initiated by adding50 μl solution of peptide substrate (1 μM) in reaction buffer (5 mMBolt™, 50 mM Tris, pH 8.0, 0.75 M Na₂SO₄). The substrate was CBR1_488 orCovidyte™ TF670, which results in fluorescence at 488 nm or 670 nm,respectively, upon cleavage by 3CL protease. Fluorescence was monitoredon Synergy™ HT plate reader (BioTek), with emission/excitation at485/525 nm or 590/645 nm, respectively. The indicated concentrations arethose of shikonin as reported by the manufacturer (i.e., assumesoriginal extract contains 30% shikonin).

As shown in Table 2, the extract inhibited 3CL protease, with the IC₅₀being between 1.25 and 2.5 μM for the assay with detection at 488 nm,and between 5 and 10 μM for the assay with detection at 670 nm.

TABLE 2 Enzymatic activity of SARS-CoV-2 3CL protease in presence ofvarious amounts of Arnebia euchroma extract, as determined byfluorescent assays at 488 and 670 nm (nominal shikonin concentrationbased on extract having 30% shikonin as reported by manufacturer)Nominal Activity shikonin (RFU/minute) concentration 488 nm assay 670 nmassay 20 μM 16 137 10 μM 27 153 5 μM 55 259 2.5 μM 125 315 1.25 μM 345398 0 μM 604 419 Blank 15 18

These results indicate that Arnebia euchroma extract comprises shikoninderivatives which inhibit 3CL protease of SARS-CoV-2, and which may beeven more potent than shikonin.

Example 9 Preparation of Exemplary Enteric Coated Tablet Formulations

Tablet cores were prepared as follows: 30 mg per tablet microcrystallinecellulose (Avicel® 101), 10 mg per tablet colloidal silicon dioxide(Aerosil® 200, obtained from Evonik), 250 mg per tablet phosphatidylserine, and 275 mg per tablet shikonin at about 95% purity (obtainedfrom Henan) were mixed by wet granulation, with the aid of 40 mgethanol. 200 mg per tablet mannitol DC (obtained from Merck), 10 mg pertablet colloidal silicon dioxide (Aerosil® 200), 100 mg per tabletmicrocrystalline cellulose (Avicel® 102, obtained from Mingtai), 20 mgper tablet crospovidone (Kollidon® CL), and 10 mg per tablet magnesiumstearate were then mixed by dry granulation. The tablet cores wereformed by tablet press using a Dio/Punch set no. 3542×7480/19.5×9 mm,oval standard, capsule shape.

The tablet cores were then coated by a composition comprising 28 gramsEudragit® L100 enteric polymer, 5.6 grams triethyl citrate, 193.2 grams1-propanol, 113.2 grams acetone, 1.5 gram Red Ponceau, to obtain red,smoothly coated tablets, as depicted in FIG. 4 .

Additional tablets were prepared as described hereinabove, with theingredients in the wet granulation and dry granulation stages being asdescribed below in Table 3.

TABLE 3 Exemplary tablet core compositions Ingredients (mg per tablet)Composition A Composition B Composition C Wet granulationMicrocrystalline cellulose (Avicel ® 101) 200 200 200 20%Phosphatidylserine 100 100 100 Phosphatidylcholine (Phospholipon ® 90)78 78 78 Shikonin (~95% purity) 112 112 112 Poloxamer 407 (Spectrum) — —45 Colloidal SiO₂ (Aerosil ® 200) — — 20 Vitamin E TPGS — 40 — Ethanol80 40 40 Dry granulation Colloidal SiO₂ (Aerosil ® 200) 10 10 10Mannitol DC 260 260 255 Microcrystalline cellulose (Avicel ® 102) 100100 100 Crospovidone (Kollidon ® CL) 30 30 30 Magnesium stearate 10 1010

The tablet cores were formed by tablet press using a Dio/Punch set no.3543×6496/16×9 mm, oval standard, capsule shape.

The tablet cores were then coated by the coating composition describedhereinabove.

Tablets (with about 250 mg shikonin) were analyzed by HPLC and UVspectroscopy and Dissolution apparatus II (USP). Tablets were exposed tosimulated gastric fluid (pH 1.0-1.2, 0.1 N HCl) for 120 minutes,followed by simulated intestinal fluid (pH 6.8, 0.1 SLS in buffer) for120 minutes.

As shown in FIG. 5 , almost none of the shikonin was released undersimulated gastric conditions over the course of 2 hours, whereas about75% of the shikonin was released within 2 hours under simulatedintestinal conditions.

The dissolution of tablets prepared with Compositions A, B and C (asdescribed in Table 3) was analyzed as described hereinabove.

As shown in Table 4, tablets formed from each of Compositions A, B and Creleased almost of the shikonin therein upon exposure to simulatedintestinal conditions, while releasing almost none of the shikonin undersimulated gastric conditions. Tablets of Composition C (with about 5%poloxamer) dissolved particularly rapidly, whereas tablets ofCompositions A and B dissolved more gradually.

TABLE 4 Percentage of shikonin released from exemplary tablets uponexposure for 2 hours to simulated gastric conditions (pH 1.0) followedby exposure for 6 hours to simulated intestinal conditions (pH 6.8) TimeComposition A Composition B Composition C (hours) pH (% release) (%release) (% release) 0 1.0 0.0 0.0 0.0 2 1.0 0.0 0.2 0.0 2.5 6.8 16.00.4 3.6 3 6.8 38.0 22.4 73.0 4 6.8 71.7 55.7 90.0 5 6.8 79.5 78.9 91.0 66.8 82.2 86.2 91.0 8 6.8 89.1 93.8 91.0

These results indicate that the tablets release shikonin in an effectiveand highly controlled manner, and that dissolution rate may becontrolled using appropriate excipients, such as poloxamer.

Example 10 Preparation of Exemplary Capsule Formulations

Phospholipid-containing, phytosome-like compositions comprising aLithospermum erythrorhizon extract were prepared as described in Table5, and used to fill size 0 capsules (by semi-automatic capsule fillingmachine). Ingredients were mixed by wet granulation, and in some casesby dry granulation, as detailed in Table 5. The weight of the capsulecontent was selected for optimal flow of granules into the capsules.

TABLE 5 Exemplary capsule content compositions Ingredients (mg percapsule) and granulation technique (wet or dry) Composition CompositionComposition Ingredient I II III Microcrystalline cellulose 200 152 125(Avicel ® 101) (wet) (wet) (wet) 20% Phosphatidylserine 100 100 100(wet) (wet) (dry) Phosphatidylcholine 78 60 50 (Phospholipon ® 90) (wet)(wet) (wet) Shikonin (~95% purity) 107 115 130 (wet) (wet) (wet) L.erythrorhizon extract 100 100 100 (wet) (wet) (dry) Poloxamer 407(Spectrum) 45 30 25 (wet) (wet) (wet) Colloidal SiO₂ 20 20 20 (Aerosil ®200) (wet) (wet) (wet) Ethanol 40 100 80

The dissolution of capsules filled with Compositions I, II and III (asdescribed in Table 5) were analyzed according to procedures such asdescribed in Example 9.

As shown in Table 6, Composition I exhibited slow release (possiblyassociated with an effect of the plant extract), whereas Compositions IIand III exhibited full release after about 1-2 hours in simulatedintestinal conditions. No significant release was observed undersimulated gastric conditions.

TABLE 6 Percentage of shikonin released from exemplary capsules uponexposure for 2 hours to simulated gastric conditions (pH 1.0) followedby exposure for 6 hours to simulated intestinal conditions (pH 6.8) TimeComposition I Composition II Composition III (hours) pH (% release) (%release) (% release) 0 1 0.0 0.0 0.0 2 1.0 2.0 2.3 3.5 2.5 6.8 19.3 31.062.1 3 6.8 28.0 64.9 93.1 4 6.8 37.2 91.4 102.4 5 6.8 38.8 91.3 102.9 66.8 39.3 91.5 102.7 8 6.8 89.1 93.8 91.0

These results indicate that capsules can release shikonin in aneffective and highly controlled manner.

Example 11 Exemplary Formulations with Additional Active Agents

A composition is prepared according to procedures as described in anyone of Example 1, 4, 5 and 6, except that the composition furtherincludes at least one additional active agent with a 3C proteaseinhibitor activity (e.g., 2-hexanoyl-4,6-dichloro-5-methoxyresorcinol,a.k.a. “DIFF-1”) and/or an anti-inflammatory activity, such as:

an IL6 and/or IL17 inhibitor, optionally one or more flavonoid (e.g.,deoxykaempferol or quercetin), substituted flavonoid (e.g.,epigallocatechin gallate (EGCG) and/or quercitrin), stilbenoid (e.g.,resveratrol and/or O-trimethyl-resveratrol), polyphenol (e.g., ellagicacid), curcuminoid (e.g., curcumin), berberine, celastrol, and/or plantextract (e.g., a Boswellia extract and/or henna (Lawsonia inermis)extract); and/or

an elastase inhibitor, optionally N-acetylcysteine and/or one or morecompounds depicted in FIG. 3 .

The composition is optionally tested for activity in vitro (e.g.,according to procedures described in Example 2 or 8); and/or in aclinical setting (e.g., according to procedures described in Example 3or 7). The composition may be assessed for synergistic activity bycomparison with the activity of naphthoquinone (e.g., shikonin of aderivative thereof) alone and the abovementioned additional activeagent(s) without the naphthoquinone.

Alternatively, a composition as described in Example 1 is used incombination with one or more compositions that include one or more ofthe additional active agents described above.

The two or more compositions are tested for activity in vitro (e.g.,according to procedures described in Example 2 or 8), by contacting the3CL protease with the compositions, either simultaneously orsequentially; and/or in a clinical setting (e.g., according toprocedures described in Example 3 or 7), by administering the two ormore compositions to a patient either simultaneously or sequentially.The combination therapy may be assessed for synergistic activity bycomparison with the activity of the naphthoquinone alone and theabovementioned additional active agent(s) without the naphthoquinone.

Example 12 Liposomal Formulations

Liposomes are prepared and loaded with shikonin or a derivative thereof(e.g., derived from a plant extract) according to any suitable techniqueknown in the art. The amount of liposomes is optionally such that aweight of the liposome lipids (e.g., lecithin, phosphatidylcholine,phosphatidylserine and/or PEGylated lipids) is from 2% to 100% of theweight of the shikonin (or derivative thereof) in the composition (e.g.,from 5 to 500 mg liposome lipids). The liposomal formulation optionallyfurther comprises one or more additional active agents such as describedin Example 8.

The liposomes are optionally formulated using procedures such asdescribed hereinabove, e.g., by placing a liquid composition comprisingliposomes in a capsule shell.

To form a “phytosome” formulation, phosphatidylcholine and/orphosphatidylserine and a plant extract described herein (which comprisesshikonin or a derivative thereof) are optionally dissolved and mixedrigorously in ethanol, and then filtered, followed by solventevaporation (e.g., at room temperature). The obtained solid is ground togranule size of less than 80 microns, and then homogenized.

The liposomal composition is optionally tested for activity in vitro(e.g., according to procedures described in Example 2 or 8); and/or in aclinical setting (e.g., according to procedures described in Example 3or 7). The effect of the liposomes may be determined by comparison withthe activity of a corresponding composition without liposomes.

Example 13 Compositions Comprising Additional Naphthoquinone Derivatives

A composition is prepared according to procedures described in Example1, 4, 5, 6, 11 and/or 12, except that instead of (or in addition to) theactive ingredient described hereinabove, a related compound, such asalkannin (e.g., purified alkannin from a synthetic or natural source, oralkannin which is part of an extract of a plant, such as Alkannatinctoria another member of the borage family), deoxyshikonin or anester (e.g., acetate, isobutyrate, isovalerate 2-methyl-butyrate,β-hydroxyisovalerate, or β,β-dimethylacrylate ester) of shikonin oralkannin is used.

The composition is optionally tested for activity in vitro (e.g.,according to procedures described in Example 2 or 8); and/or in aclinical setting (e.g., according to procedures described in Example 3or 7).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

It is the intent of the applicant(s) that all publications, patents andpatent applications referred to in this specification are to beincorporated in their entirety by reference into the specification, asif each individual publication, patent or patent application wasspecifically and individually noted when referenced that it is to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting. In addition, anypriority document(s) of this application is/are hereby incorporatedherein by reference in its/their entirety.

What is claimed is:
 1. A method of treating a coronavirus infection in asubject in need thereof, the method comprising administering to thesubject a compound represented by Formula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring, and a) wherein the compound ispart of a composition which further comprises phospholipids, and aweight ratio of said phospholipids to the compound in said compositionis in a range of from 10:1 to 1:10, and/or b) wherein the compound ispart of a composition which further comprises liposomes; therebytreating the coronavirus infection.
 2. The method according to claim 1,wherein at least one, or each, of R₃ and R₆ is OH.
 3. The methodaccording to claim 1, wherein R₁ is represented by:

wherein R′₁, R″₁, and R₇ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heteroalicyclic, and carbonyl.
 4. The method according to claim 3,wherein R₇ is a saccharide moiety or a peptide moiety, or wherein R₇ ishydrogen or carbonyl.
 5. The method according to claim 1, wherein thecompound is shikonin or a glycoside of shikonin, or an ester thereof. 6.The method according to claim 1, wherein said treatment is of a subjectin which inhibiting inflammation would be beneficial.
 7. The methodaccording to claim 1, wherein said treatment further comprisesadministering to the subject at least one additional active agentselected from the group consisting of a vitamin, N-acetyl cysteine, ananticoagulant, an anti-inflammatory agent, an antipyretic agent, anantiviral agent, and a protease inhibitor.
 8. A method of treating asubject in need thereof, the method comprising administering to thesubject a compound represented by Formula I:

wherein R₁-R₆ are each independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano,nitro, azide, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, a ureagroup, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, S-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide,and amino, or alternatively, R₁ and R₂ together form a five- orsix-membered aromatic or aliphatic ring and a) wherein said compound ispart of a composition which further comprises phospholipids, and aweight ratio of said phospholipids to said compound in said compositionis in a range of from 10:1 to 1:10, and/or b) wherein said compound ispart of a composition which further comprises liposomes; therebytreating the subject.
 9. The method of claim 8, wherein at least one oreach of R₃ and R₆ is OH.
 10. The method of claim 8, wherein R₁ isrepresented by the formula:

wherein R′₁ and R″₁ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, andheteroalicyclic.
 11. The method of claim 10, wherein R′₁ is alkenyl. 12.The method of claim 10, wherein R₇ is a saccharide moiety or a peptidemoiety or wherein R₇ is hydrogen or carbonyl.
 13. The method of claim 8,wherein the compound is shikonin or a glycoside of shikonin, or an esterthereof.
 14. The method of claim 8, wherein the subject will benefitfrom: i) inhibiting of an activity of a 3CL protease activity in saidsubject, ii) inhibiting inflammation in said subject, and iii)inhibiting autophagy in said subject.
 15. The method of claim 8, furthercomprising administering to the subject at least one additional activeagent selected from the group consisting of a vitamin, N-acetylcysteine, an anticoagulant, an anti-inflammatory agent, an antipyreticagent, an antiviral agent, and a protease inhibitor.